1. Field of the Invention
The present invention relates to disposable reagent-containing elements which can be used in conjunction with an electronic instrument for performing diagnostic assays (medical diagnostics). It also relates to a method for performing diagnostic biological assays employing the use of a disposable reagent-containing element.
2. Discussion of the Background
Many analytical techniques have been developed for chemical, biochemical and biological assays. Procedures that use a discrete fluid sample for the analysis of a single analyte are traditionally characterized as wet chemical techniques or dry chemical techniques. In recent years both types of techniques have been automated to reduce costs and simplify procedures. Wet chemical methods, typified by the TECHNICON.RTM. autoanalyzers, utilize batches of reagent solutions, pumps and fluid controls, coupled with conventional sensors such as radiometric (e.g.: fluorescent, colorimetric, or nephelometric) electrochemical (e.g.: conductometric, polarographic, or potentiometric) and others, such as ultrasonic, etc. sensors. These techniques are characterized by large equipment, and are generally expensive. They are complicated, and require a skilled operator.
Decentralized testing, particularly in medical applications, has been achieved with a variety of simpler systems often based on cuvettes for optical determination but sometimes based on dry chemistry-based "reagent strip" technology. Generally, a reagent strip is an absorbent structure containing a reagent which self-meters an applied sample and develops or changes color to indicate the extent of reaction. Although self metering, some reagent strips, however, require pipetting of sample to achieve maximum accuracy and precision. A reagent strip is employed either by itself or in conjunction with a simple instrument to read the color intensity or hue and translate the results into a numerical value which is displayed. Unlike a cuvette or test tube, mixing or convection cannot be sustained in a reagent strip after the sample has entered, and once having entered, the sample cannot be removed from within without destroying the integrity of the strip. In one application, reagent strip technology is used extensively in the home by diabetic patients who test themselves daily to determine blood sugar levels.
In a more general example of "reagent strip" technology, Eastman Kodak has introduced a system of dry chemistry, claiming to overcome many of the traditional weaknesses of dry chemical methods. The Kodak technique utilizes flat, multi-layered sheets arranged in sequence. The top layer receives a liquid sample which passes downward undergoing separation and reactions in a pre-arranged sequence. The sheet is designed to accept a small volume of liquid and distribute it uniformly over a reproducible area. The area is less than the total area of the multi-laminar sheet. Each layer of the sheet is essentially homogenous in a direction parallel to the surface. Once the sample has spread radially (a rapid process), the components of the liquid can move downward at rates that are essentially the same in any plane that is parallel to the surface. In this way uniform reactions, filtrations, etc. can occur.
The analyte is detected in the multi-layered sheets by radiometric or electrochemical methods which are carried out in a thermostated environment. This permits the use of kinetic and static measurements to detect analyte concentrations in a liquid sample.
Radiation is caused to enter this assembly in a path which is traverse to the several layers. The radiation is modified by the analyte or by a component or product of the analyte. For example, the exciting radiation may be partially absorbed by the analyte or by a component or product of the analyte. The modified radiation may be reflected back transversely through the laminar assembly, typically from a reflective layer, adjacent or nearly adjacent to a thin layer where color is formed. Thus, reflectance can be monitored (as opposed to transmission only through the color producing layer). Reflectance, as expressed by Kubelka-Monk theory, consists of optical density absorbance and scattering components and is more sensitive than transverse colorimetry through a thin, turbid layer. Reflectance, however, may be a more difficult technique to standardize and to interpret data from than colorimetry.
Conventional colorimetry has not been practiced with reagent strips because the color producing layers are generally thin and not transparent. The path of the exciting radiation is thus very short (with large light losses due to opacity) and is determined by the thickness of the layer in which the exciting radiation encounters the substance which is excited. Since this dimension must be very small to permit rapid measurement, e.g., 10 .mu.m to 100 .mu.m, the degree of modification of the exciting radiation is quite small. This limits the applicability of this technique to analyses in which the analyte (or the product of the analyte) interacts very strongly with the exciting radiation, otherwise a very sensitive detecting apparatus has to be used. This method has been shown to be useful for measuring analytes in blood that exist at relatively high concentrations, e.g., glucose, BUN, cholesterol and albumin.
Other analytical methods have been developed that utilize rapidly reversable chemical reactions to continuously monitor analyte concentrations in biological fluids, or industrial effluent streams, or ponds, lakes and streams. For example, several methods have been proposed to measure the oxygen level in blood of critically ill patients.
Reagent strip technology, however, possesses salient drawbacks and limitations. For example, once a sample is added to a reagent strip and permeates the porous structure of the strip, the sample cannot be removed or washed out without destroying the integrity of the strip. For example, immunoassays are extremely difficult to perform with reagent strips, in part because separation of free and bound antigen (or antibody) molecules from a mixture of both cannot be readily achieved in a conventional porous or layered structure. This limits possible immunoassay applications to certain special cases of reactions, for example certain homogenous reaction sequences. Incubation with mixing, a step common to a variety of assays, cannot be performed easily in conventional reagent strip formats since they rely on diffusion and initial capillary action only for mixing.
Technologies have not yet been developed to cause or to control forced convection for a specified period of time within the porous structure of a reagent strip after the sample has entered and permeated the porous strip structure. In conventional reagent strips, the strip is an absorbant matrix in which mixing is extremely difficult and limited. In addition, reagent strips almost exclusively use reflectance as a photometric method to quantitatively determine the extent of a color reaction. There are no reagent strips known to the inventor which can be read via light transmission/absorbance colorimetry, nephelometry, fluorescence, chemiluminescence, or evanescent wave technology. Fluorescence measurement is possible in reagent strips, but difficult to achieve. Electrochemistry has been used successfully.
As discussed above, a large number of types of medical tests are carried out by trained medical laboratory personnel. These tests must be performed accurately and reproducibly with a minimum amount of error since they are used as aids in diagnosing and treating medical ailments. To aid laboratory personnel in performing these tests accurately on a large number of samples in a relatively short period of time, auxiliary equipment, which is often expensive, is frequently used. Most of these tests are performed on a macro scale and thus require considerable quantities of both sample and reactants. They also require varying degrees of sample preparation. These and other reasons are major contributors to the generally relatively expensive nature and sources of error in medical diagnostic tests performed on body fluids.
Improvements have been made in some medical tests. For example, the reagent strip technology discussed previously simplifies medical tests, minimizes the required quantities of sample and/or reactants, can minimize possible sources of error, and lower costs. Various types of medical tests, however, have been difficult to perform accurately and economically on either a macro or a micro scale. In this respect, medical tests which require rapid and thorough mixing of reagents with a sample to provide a clearly defined starting point, an accurate measurement of reaction time, and a clear determination of the reaction endpoint, have been particularly difficult to perform with simple and inexpensive devices and have been plagued with inaccuracies resulting from errors in measurement and manipulation.
Once such type of test is the blood prothrombin time test ("PT" hereinafter). This test measures the time required to form a blood clot (via extrinsic and intrinsic blood coagulation physiologic pathways).
Coagulation assays, in general, are used for a variety of reasons. They are principally used for monitoring patients receiving anticoagulant therapy. The most frequently performed coagulation assay is PT. Prothrombin time determinations are used to monitor patients receiving oral anticoagulants such as warfarin. An accurate monitoring of coagulation in these patients is important to prevent recurrent clotting (thrombosis) and to keep the coagulation mechanism sufficiently active to prevent internal bleeding. Prothrombin time testing provides information to permit better drug control to be achieved through the regulation of drug dosage.
In conventional practice, PT assays are performed by the addition of a liquid reagent to a plasma sample. The reagents are typically supplied in dried form and consist primarily of thromboplastin and calcium chloride. The dried reagent is reconstituted before use by addition of a measured amount of distilled water. The reagent is thermally sensitive, and refrigeration prior to use is required. The shelf life of the reagent in dried form is from one to two years. However, when it is reconstituted the reagent is considerably more labile and must be used within a few hours or discarded. In some cases reconstituted reagents can be kept for a few days under refrigeration.
Prothrombin time assays are performed by mixing sample and reagent at 37.degree. C., and monitoring the progress of the reaction until a perceptible clot (or "gel clot") is detected. The development of a gel clot is the end point of the reaction. This end point may be detected in various ways; by viscosity change, by electrode reaction; and, most commonly, by photometric means. The test result is generally compared to a result using a normal (control) plasma.
Before performing the test, the blood sample is collected in the tube or syringe containing anti-coagulant (citrate). The blood sample is centrifuged, and the plasma separated (e.g., by decantation) from the red blood cells. A measured quantity (usually 0.1 ml) of plasma is pipetted into the reaction vessel or cuvet. A measured amount of reagent is then added manually via pipette or automatically by means of other volumetric delivery systems capable of metering a known, preset quantity of reagent. Alternatively, the sample can be added to the reagent directly. Typically, 0.2 ml of reagent is employed. The addition of the reagent initiates the reaction.
Some PT kits for use in the home are known. For example, McCormick (U.S. Pat. No. 3,233,975) discloses a prothrombin reaction chamber. The chamber is constructed of a transparent material so that the progress of the reaction can be visually monitored. To perform a blood prothrombin time test with this chamber, one adds sequentially a measured volume of a prepared blood sample and a measured volume of an aqueous solution of reagent to the chamber. The chamber is then manually agitated, and the progress of the reaction visually monitored and timed with a stop watch.
This prothrombin reaction chamber, however, suffers from numerous disadvantages. For the prothrombin test to be performed with this reaction chamber, a prepared blood sample is used. Thus sample manipulation is required. A specific volume of the prepared blood sample must be added to the chamber. The measurements involved in obtaining this specific volume of prepared blood sample contribute inaccurate results and considerable labor.
This reaction chamber also requires the preparation of a solution containing the reagent(s). The precise measurement of the amounts of materials and water to be combined in preparing the reagent solution introduces another additional source of error. The measurement of the quantity of reagent solution to be added to the chamber provides a further source of error. Moreover, as discussed above, having to use a reagent solution is undesirable because of potential stability problems. If the reagent solution is not used within a few hours, the solution must be discarded.
McCormick's prothrombin reaction chamber is based on the visual observation of the reaction to measure clotting time. It does not permit accurate monitoring of sample mixing with the reagent(s), accurate determination of reaction starting point (which is as important as the end point when reaction time is being measured), or accurate determination of reaction end point.
Accordingly there is a strongly felt need for a facile and accurate method for the performance of biological assays, e.g., in medical application. Such method should be based on a minimum number of manipulations of either a sample or reagent.
Ideally it should require no sample or reagent-containing solution preparation. It should minimize problems associated with reagent instability and optimize accuracy. It should permit effective mixing of sample and reagent. It should permit sample manipulation. It should require only a very small amount of sample. And it should be able to perform automatic treatments of the sample, e.g., separate red blood cells from plasma in blood. This method should be based on a simple and inexpensive reagent-containing element.